Therapeutic retroviral vectors for gene therapy

ABSTRACT

Retroviral gene therapy vectors that are optimized for erythroid specific expression and treatment of hemoglobinopathic conditions are disclosed.

PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/150,785, filed on Jun. 9, 2005, which is a continuation ofInternational Patent Application Ser. No. PCT/US03/039526, filed on Dec.11, 2003, which claims priority to U.S. Provisional Application No.60/433,321, filed on Dec. 13, 2002; U.S. Provisional Application No.60/475,822, filed on Jun. 4, 2003; and U.S. Provisional Application No.60/513,312, filed on Oct. 21, 2003. The contents of each of theaforementioned applications are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

Hemoglobinopathies encompass a number of anemias of genetic origin inwhich there is decreased production and/or increased destruction(hemolysis) of red blood cells (RBCs). The blood of normal adult humanscontains hemoglobin (designated as HbA) which contains two pairs ofpolypeptide chains designated alpha and beta. Fetal hemoglobin (HbF),which produces normal RBCs, is present at birth, but the proportion ofHbF decreases during the first months of life and the blood of a normaladult contains only about 2% HbF. There are genetic defects which resultin the production by the body of abnormal hemoglobins with a concomitantimpaired ability to maintain oxygen concentration. Among thesegenetically derived anemias are included thalassemia, Cooley's Diseaseand sickle cell disease.

Sickle cell disease (SCD) is one of the most prevalent autosomalrecessive diseases worldwide. SCD became the first genetic disorder forwhich a causative mutation was identified at the molecular level: thesubstitution of valine for glutamic acid in human β^(A)-globin codon 6(Ingram (1957) Nature, 180:326). In homozygotes the abnormal hemoglobin(Hb) [HbS (α₂β^(S) ₂)] polymerizes in long fibers upon deoxygenationwithin red blood cells (RBCs), which become deformed or “sickled,”rigid, and adhesive, thereby triggering microcirculation occlusion,anemia, infarction, and organ damage (Stamatoyannopoulos, et al. (eds)(1994) The Molecular Basis of Blood Diseases, Saunders, Philadelphia,ed. 2; 207-256; Nagel, et al. (2001) Disorders of Hemoglobin, CambridgeUniv. Press, Cambridge; 711-756).

Human γ-globin is a strong inhibitor of HbS polymerization, in contrastto human β^(A)-globin, which is effective only at very highconcentrations (Bookchin et al. (1971) J. Mol. Biol. 60:263). Hence,gene therapy of SCD was proposed by means of forced expression ofγ-globin or γ/β hybrids in adult RBCs after gene transfer tohematopoietic stem cells (HSCs) (McCune et al. (1994) PNAS USA 91:9852;Takekoshi et al. (1995) PNAS USA 92:3014; Miller et al. (1994) PNAS USA91:10183; Emery et al. (1999) Hum. Gene Ther. 10:877; Rubin et al.(2000) Blood 95: 3242; Sabatino et al. (2000) PNAS USA 97:13294; Blouinet al. (2000) Nat. Med. 6:177).

Although the discovery of the human β-globin locus control region (LCR)held promise to achieve high globin gene expression levels (Tuan et al.(1985) PNAS USA 82:6384; Grosveld et al. (1987) Cell 51:975), the stabletransfer of murine onco-retroviral vectors encompassing minimal coreelements of the LCR proved especially challenging (Gelinas et al. (1992)Bone Marrow Transplant 9:157; Chang et al. (1992) PNAS USA 89:3107;Plavec et al. (1993) Blood 81:1384; Leboulch et al. (1994) EMBO J13:3065; Sadelain et al. (1995) PNAS USA 92:6728; Raftopoulos et al.(1997) Blood 90:3414; Kalberer et al. (2000) PNAS USA 97:5411). To allowthe transfer of larger LCR and globin gene sequences, one proposal wasthe use of RNA splicing and export controlling elements that include theRev/R responsive element (RRE) components of human immunodeficiencyvirus (HIV) (Alkan et al. (31 May 2000) paper presented at the 3rdAmerican Society of Gene Therapy, Denver, CO), and an RRE-bearingHIV-based lentiviral vector which had resulted in substantialamelioration of β-thalassemia in transplanted mice (May et al. (2000)Nature 406:82). This approach was not sufficient for completecorrection, however, as gene expression remained heterocellular, and theamount of human β^(A)-globin found incorporated in Hb tetramers in anonthalassemic background was unlikely to be successful therapy for SCD(May et al., supra). Accordingly, there remains a need for a genetherapy approach which can successfully treat SCD and otherhemoglobinopathies.

SUMMARY OF THE INVENTION

The present invention provides improved compositions and methods forachieving gene therapy in hematopoietic cells and hematopoieticprecursor cells, including erythrocytes, erythroid progenitors, andembryonic stem cells. The invention further provides improved genetherapy methods for treating hematopoietic-related disorders.

In one embodiment, the invention provides an improved gene therapyvector optimized to express high levels of one or more therapeuticproteins in erythroid cells or erythroid precursor cells. In aparticular embodiment, the vector comprises an optimized retroviralvector which expresses one or more antisickling proteins at therapeuticlevels in order to treat hemoglobinopathies. Retroviral vectors,including lentiviral vectors, employed in the gene delivery system ofthe present invention are highly efficient at infecting and integratingin a non-toxic manner into the genome of erythroid cells, andmaintaining therapeutic levels of erythroid-specific gene expression. Ina particular embodiment, the retroviral vector of the inventioncomprises a left (5′) retroviral LTR; a retroviral export element,optionally a lentiviral reverse response element (RRE); a promoter, oractive portion thereof, and a locus control region (LCR), or activeportion thereof, operably linked to a gene of interest; and a right (3′)retroviral LTR. The retroviral vector of the invention can furthercomprise a central polypurine tract/DNA flap (cPPT/FLAP), including, forexample, a cPPT/FLAP from HIV-1. In one embodiment, the retrovirus is alentivirus, including, for example, HIV. In another embodiment, thepromoter of the 5′ LTR is replaced with a heterologous promoter,including, for example, cytomegalovirus (CMV) promoter,

Retroviral vectors, including lentiviral vectors, of the inventionfurther comprise a gene of interest, including, for example, a globingene or a gene which encodes an antisickling protein. In one embodiment,the globin gene expressed in the retroviral vector of the invention isβ-globin, δ-globin, or γ-globin. In another embodiment, the humanβ-globin gene is the wild type human β-globin gene or human β^(A)-globingene. In another embodiment, the human β-globin gene comprises one ormore deletions of intron sequences or is a mutated human β-globin geneencoding at least one antisickling amino acid residue. Antisicklingamino acids can be derived from human δ-globin or human γ-globin. Inanother embodiment, the mutated human β-globin gene encodes a threonineto glutamine mutation at codon 87 (β^(A-T87Q)).

Retroviral vectors, including lentiviral vectors, of the invention canbe used in gene therapy, including for the treatment ofhemoglobinopathies. The invention also includes host cells comprising,e.g., transfected with, the vectors of the invention. In one embodiment,the host cell is an embryonic stem cell, a somatic stem cell, or aprogenitor cell.

In other embodiments, the invention provides methods for using theforegoing optimized vectors to achieve stable, high levels of geneexpression in erythroid cells, e.g., in order to treaterythroid-specific diseases. In a particular embodiment, the genetherapy vectors are used to treat hemoglobinopathies, including, forexample, sickle cell disease (SCD). In another embodiment, the genetherapy vectors are used for treatment of thalassemias, including, butnot limited to, β-thalassemia.

In yet other embodiments, the invention provides a self-inactivating(SIN) retroviral vector comprising a left (5′) retroviral LTRaretroviral export element, optionally a lentiviral reverse responseelement (RRE); a promoter, or active portion thereof, and a locuscontrol region (LCR), or active portion thereof, operably linked to agene of interest; and a right (3′) retroviral LTR, wherein the U5 regionof the left (5′) LTR, the right (3′) LTR, or both the left and rightLTRs are modified to replace all or a portion of the region with anideal poly(A) sequence and the U3 region of the left (5′) long terminalrepeat (LTR), the right (3′) LTR, or both the left and right LTRs aremodified to include one or more insulator elements. In one embodimentthe U3 region is modified by deleting a fragment of the U3 region andreplacing it with an insulator element. In yet another embodiment, theU5 region of the right (3′) LTR is modified by deleting the U5 regionand replacing it with a DNA sequence, for example an ideal poly(A)sequence. In yet another embodiment, the vector further comprises acentral polypurine tract/DNA flap (cPPT/FLAP). In still anotherembodiment, the vector comprises an insulator element comprising aninsulator from an α-globin locus, including, for example, chicken HS4

In another embodiment of the invention, the vector includes a nucleicacid cassette comprising a suicide gene operably linked to a promoter.In a particular embodiment, the suicide gene is HSV thymidine kinase(HSV-Tk). The vector can also include a nucleic acid cassette comprisinga gene for in vivo selection of the cell, such as a gene for in vivoselection, e.g., a methylguanine methyltransferase (MGMT) gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a graphically shows that β^(A-T87Q) and HbF are potent inhibitorsof HbS polymerization in vitro in contrast to HbA. FIG. 1 b shows aSouthern blot analysis for proviral stability. Lane 1, NIH 3T3 negativecontrol; lanes 2-4, bone marrow, spleen and thymus DNA, respectively,from a representative C57B1/6 recipient of A-T87Q-globin transduced bonemarrow sacrificed 5 months post-transplantation; lanes 5 and 6, DNA from2 day 12 spleen colonies generated using bone marrow from the primaryC57B1/6 recipient sacrificed 5 months post-transplantation. The expected7.5 Kb proviral band and a 3.2 Kb endogenous band (EB) are marked in theleft margin. Bottom: average proviral copy number in genomic DNAisolated from blood of A-T87Q-globin transduced C57B1/6 mice 3 monthspost-transplantation (bar=SE). Quantification was performed bydensitometry and comparison to NIH3T3 cells known to contain one copy ofthe provirus. FIG. 1 c graphically depicts the β^(A-T87Q)-globinprovirus. HIV LTR, human immune deficiency type-1 virus long terminalrepeat; Ψ+, packaging signal; cPPT/flap, central polypurine tract/DNAflap; RRE, Rev-responsive element; βP, β-globin promoter (from SnaB I toCap site); ppt, polypurine tract. The 3´β-globin enhancer (up todownstream Avr II site), the 372 by IVS2 deletion, the β^(A-T87Q)mutation (ACA Thr to CAG Gln) and DNase I hypersensitive sites (HS)2(Sma I to Xba I), HS3 (Sac I to Pvu II) and HS4 (Stu I to Spe I) of theβ-globin LCR are indicated.

FIG. 2 graphically depicts results from an analysis of humanβ^(A-T87Q)-globin gene expression in C57BL/6 recipient mice 5 monthsafter transplantation. FIG. 2 a shows circulating RBCs from recipientmice were fixed permeabilized, stained with a FITC-labeled antibody thatspecifically recognizes human β-globin (Perkin-Elmer Wallac, NortonOhio), and subsequently analyzed by FACS. Top: representative mousetransplanted with mock-transduced bone marrow cells. Bottom:representative mouse transplanted with bone marrow transduced with theβ^(A-T87Q)-globin lentivirus. FIG. 2 b shows results from primerextension analysis of peripheral blood RNA. Lanes 1, 3, 5, 7, and 9:amplification with primers specific for the endogenous murine β-singleglobin mRNA generating a 53-base pair (bp) DNA fragment. Lanes 2, 4, 6,8, and 10: amplification with primers specific for the humanβ^(A-T87Q)-globin mRNA generating a 90-bp DNA fragment. Lanes 1 and 2:mock-transduced mouse. Lanes 3 and 4: transgenic control mouseexpressing 86% of human β-globin mRNA. Lanes 5 to 10: three C57BL/6recipients of β^(A-T87Q)-globin-transduced bone marrow cells (lanes 5and 6, mouse #1; 7 and 8 mouse #2; 9 and 10, mouse #3). FIG. 2 cgraphically depicts HPLC profiles of globin chains extracted from RBCsof a mock-transduced mouse (top) and a recipient of humanβ^(A-T87Q)-globin-transduced bone marrow (bottom).

FIG. 3 graphically depicts HPLC profiles of Hb extracted from RBCs ofmouse recipients of mock-transduced SAD (FIG. 3 a), mock-transduced BERK(FIG. 3 b), β^(A-T87Q)-globin-transduced SAD (FIG. 3 c), andβ^(A-T87Q)-globin-transduced BERK bone marrow cells (FIG. 3 d).

FIG. 4 shows isoelectric focusing of RBC lysates from recipient mice 3months post-transplantation showing the expected species of Hb. Lanes 1and 2, blood deriving from SAD transplanted marrow; lanes 3 and 4, bloodderiving from BERK transplanted marrow; lanes 1 and 3, mocktransduction; lanes 2 and 4, transduction with β^(A-T87Q)-globinlentivirus. α^(M), mouse α-globin; α^(H), human α-globin; β^(SIN), mousesingle β-globin; β^(SAD), human SAD β-globin; β^(S), human sickleβ-globin; β^(A-T87Q), human β^(A-T87Q) globin.

FIG. 5 shows correction of SCD pathology. FIG. 5 a shows Nomarski opticsmicroscopy of RBCs from mice transplanted with either (top) mock- or(bottom) β^(A-T87Q)-globin lentivirus-transduced BERK bone marrow cellsunder 5% pO₂ 3 months after transplantation. FIG. 5 b showsquantification of the percentage of sickle RBCs from recipients ofmock-transduced and β^(A-T87Q)-globin-transduced BERK or SAD bone marrowunder 5% or 13% oxygen conditions, respectively. Error bars indicate SE;*, P=0.01; †, P =0.03. FIG. 5 c shows the relationship between log ofreciprocal delay time (dt) of HbS polymerization and Hb concentration ofRBC lysates. Time courses of Hb polymerization in lysates were performedat various concentrations by the temperature jump method. Δ, lysate fromhomozygote SS patient; ▴, lysate from an asymptomatic AS sickle celltrait patient; □, lysate from a mouse recipient of mock-transduced SADmarrow; ▪, lysate from a mouse recipient of β^(A-T87Q)-globin-transducedSAD marrow; ◯, lysate from mouse recipient of mock-transduced BERKmarrow; , lysate from a mouse recipient of β^(A-T87Q)-globin-transducedBERK marrow. FIG. 5 d shows Percoll-Larex continuous density gradientsfrom blood of recipient mice. Lane 1, density marker beads; lanes 2 and6, C57BL/6 controls; lanes 3 and 7, SAD and BERK controls, respectively;lanes 4 and 5, C57BL/6 recipients of mock-transduced orβ^(A-T87Q)-transduced SAD bone marrow, respectively; lane 8, C57BL/6recipient of β^(A-T87Q)-transduced BERK bone marrow; lane 9, transgenicBERK mouse expressing human γ-globin at ˜100% of β^(S)-globin. FIG. 5 eshows spleens from nontransplanted (1) BERK and (2) C57BL/6 mice orC57BL/6 mice transplanted with either (3) β^(A-T87Q)-transduced or (4)mock-transduced BERK bone marrow.

FIG. 6 graphically depicts a map of a vector comprising a right LTR witha doublet insulator.

FIG. 7 graphically depicts a map of the SIN vector comprising a 399deletion in the right LTR U3 region, which has been replaced by adoublet insulator, and a replacement in the U5 region with an idealpoly(A) sequence. The vector also contains a GFP fusion gene.

FIG. 8 graphically depicts a map of the SIN vector comprising a 399deletion in the right LTR U3 region, which has been replaced by adoublet insulator, and a replacement in the U5 region with an idealpoly(A) sequence. This vector also contains BGT 9 (β-globin) with adeletion in intron 2.

FIG. 9 graphically depicts a map of a vector comprising a right LTR witha 42 bp insulator.

FIG. 10 graphically depicts a map of the SIN vector comprising a 399deletion in the right LTR U3 region, which has been replaced by a 42 bpinsulator, and a replacement in the U5 region with an ideal poly(A)sequence. The vector also contains a GFP fusion gene.

FIG. 11 graphically depicts a map of the SIN vector comprising a 399deletion in the right LTR U3 region, which has been replaced by a 42 bpinsulator, and a replacement in the U5 region with an ideal poly(A)sequence. This vector also contains BGT 9 (β-globin) with a deletion inintron 2.

FIG. 12 shows the nucleotide sequence of oligo OHPV #460/461, includingthe positions of the Cla I, Xba I, and Pvu II restriction sites, and the42 bp insulator.

FIG. 13 graphically depicts the SIN vector and titer analysis for theSIN vector. FIG. 13 a graphically depicts the organization of the SINvector, including the modifications to the LTR. FIG. 13 b shows adetailed schematic of the modification, wherein the U3 region of the LTRis replaced with a cHS4 insulator and the U5 region is replaced with anideal polyA sequence. FIG. 13 c shows a Southern blot which examines thetiter for the insulator SIN vector containing β-globin.

FIG. 14 shows a diagram of a lentiviral vector containing the humanβ-globin gene and results demonstrating successful expression of thehuman β-globin gene in mice. FIG. 14A graphically depicts the the humanβ-globin (β^(A)) lentiviral vector. FIG. 14B shows the proportion ofperipheral blood RBCs expressing human β-globin, as assessed by FACSafter staining the cells with an antibody specific for human β-globin.FIG. 14C shows expression of human β globin in red blood cells (RBCs) ofreconstituted mice. The upper left of FIG. 14C shows FACS analysis ofRBCs from a representative recipient of lenti-GFP-virus-transduced THALbone marrow cells, where RBCs from an unmanipulated mouse were used as anegative control. The upper right and lower left of FIG. 14C showresults from FACS analysis of RBCs from a representative recipient oflenti-β globin-virus-transduced THAL bone marrow cells at 2 and 7 monthsafter transplantation. The lower right of FIG. 14C shows results fromFACS analysis of RBCs from one of the secondary recipients transplantedwith bone marrow cells from a primary donor at 6 months aftertransplantation.

FIG. 15 shows improvement of hematological parameters in THAL mice whoreceived lenti-β globin-transduced THAL bone marrow cells. Results shownare the mean±SD for untransplanted, control THAL mice (n=5), normalunmanipulated B6 mice (n=5), and THAL mice transplanted 6 monthspreviously with lenti-β globin-transduced cells (n=8) or controllenti-GFP-transduced cells (n=6). Changes in all hematologic parametersseen in THAL recipients of lenti-β globin-transduced cells were highlysignificant in comparison with nontransplanted, or control(GFP)-transplanted THAL mice (*, P<0.001). Values for the RBC number andreticulocyte count in these corrected mice reached levels within thenormal range of control B6 mice (P=0.6 and 0.1, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved compositions and methods forgene therapy, particularly in the treatment of hemoglobinopathies. Inone embodiment, improved lentiviral vectors are use to delivertherapeutic gene products to embryonic stem cells, somatic stem cells,or hematopoietic stem cells, including, but not limited to, erythroidprogenitors. In another embodiment, improved lentiviral vectors are usedto deliver therapeutic genes to erythrocytes, thereby providingsustained, high level expression of therapeutic proteins specifically inerythroid cells. In a particular embodiment, the expression is permanent(e.g., panerythroid).

I. Definitions

As used herein, the following terms and phrases used to describe theinvention shall have the meanings provided below.

The term “retrovirus” refers to any known retrovirus (e.g., type cretroviruses, such as Moloney murine sarcoma virus (MoMSV), Harveymurine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV),gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV),spumavirus, Friend, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus(RSV)). “Retroviruses” of the invention also include human T cellleukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family ofretroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2,simian immnodeficiency virus (SW), feline immonodeficiency virus (FIV),equine immnodeficiency virus (EIV), and other classes of retroviruses.

Retroviruses are RNA viruses that utilize reverse transcriptase duringtheir replication cycle. The retroviral genomic RNA is converted intodouble-stranded DNA by reverse transcriptase. This double-stranded DNAform of the virus is capable of being integrated into the chromosome ofthe infected cell; once integrated, it is referred to as a “provirus.”The provirus serves as a template for RNA polymerase II and directs theexpression of RNA molecules which encode the structural proteins andenzymes needed to produce new viral particles.

At each end of the provirus are structures called “long terminalrepeats” or “LTRs.” The term “long terminal repeat (LTR)” refers todomains of base pairs located at the ends of retroviral DNAs which, intheir natural sequence context, are direct repeats and contain U3, R andU5 regions. LTRs generally provide functions fundamental to theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. The LTRcontains numerous regulatory signals including transcriptional controlelements, polyadenylation signals and sequences needed for replicationand integration of the viral genome. The viral LTR is divided into threeregions called U3, R and U5. The U3 region contains the enhancer andpromoter elements. The U5 region is the sequence between the primerbinding site and the R region and contains the polyadenylation sequence.The R (repeat) region is flanked by the U3 and U5 regions. The LTRcomposed of U3, R and U5 regions, appears at both the both the 5′ and 3′ends of the viral genome. In one embodiment of the invention, thepromoter within the LTR, including the 5′ LTR, is replaced with aheterologous promoter. Examples of heterologous promoters which can beused include, for example, the cytomegalovirus (CMV) promoter.

The term “lentivirus” refers to a group (or genus) of retroviruses thatgive rise to slowly developing disease. Viruses included within thisgroup include HIV (human immunodeficiency virus; including HIV type 1,and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes (i.e., T-cells).

The term “hybrid” refers to a vector, LTR or other nucleic acidcontaining both lentiviral sequences and non-lentiviral retroviralsequences.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. The term“expression vector” includes any vector, (e.g., a plasmid, cosmid orphage chromosome) containing a gene construct in a form suitable forexpression by a cell (e.g., linked to a promoter). In the presentspecification, “plasmid” and “vector” are used interchangeably, as aplasmid is a commonly used form of vector. Moreover, the invention isintended to include other vectors which serve equivalent functions.

The term “retroviral vector” refers to a vector containing structuraland functional genetic elements that are primarily derived from aretrovirus.

The term “lentiviral vector” refers to a vector containing structuraland functional genetic elements outside the LTRs that are primarilyderived from a lentivirus.

The term “self-inactivating vector” refers to vectors in which the right(3′) LTR enhancer-promoter region, know as the U3 region, has beenmodified (e.g., by deletion or substitution) to prevent viraltranscription beyond the first round of viral replication. Consequently,the vectors are capable of infecting and then integrating into the hostgenome only once, and can not be passed further. This is because theright (3′) LTR U3 region is used as a template for the left (5′) LTR U3region during viral replication and, thus, the viral transcript can notbe made without the U3 enhancer-promoter. If the viral transcript is notmade, it can not be processed or packaged into virions, hence the lifecycle of the virus ends. Accordingly, SIN vectors greatly reduce risk ofcreating unwanted replication-competent virus since the right (3′) LTRU3 region has been modified to prevent viral transcription beyond thefirst round of replication, hence eliminating the ability of the virusto be passed.

The term “TAR” refers to the “trans-activation response” genetic elementlocated in the R region of lentiviral (e.g., HIV) LTRs. This elementinteracts with the lentiviral trans-activator (tat) genetic element toenhance viral replication.

The term “R region” refers to the region within retroviral LTRsbeginning at the start of the capping group (i.e., the start oftranscription) and ending immediately prior to the start of the poly Atract. The R region is also defined as being flanked by the U3 and U5regions. The R region plays an important role during reversetranscription in permitting the transfer of nascent DNA from one end ofthe genome to the other.

The term “transfection” refers to the introduction of foreign DNA intoeukaryotic cells. Transfection may be accomplished by a variety of meansknown in the art including but not limited to calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “transduction” refers to the delivery of a gene(s) using aviral or retroviral vector by means of viral infection rather than bytransfection. In preferred embodiments, retroviral vectors aretransduced by packaging the vectors into virions prior to contact with acell. For example, an anti-HIV gene carried by a retroviral vector canbe transduced into a cell through infection and provirus integration.

The term “promoter/enhancer” refers to a segment of DNA which containssequences capable of providing both promoter and enhancer functions. Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal.The term “poly A site” or “poly A sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.”

An endogenous poly A signal is one that is found naturally at the 3′ endof the coding region of a given gene in the genome. A heterologous polyA signal is one which is one which is isolated from one gene and placed3′ of another gene.

The term “export element” refers to a cis-acting post-transcriptionalregulatory element which regulates the transport of an RNA transcriptfrom the nucleus to the cytoplasm of a cell. Examples of RNA exportelements include, but are not limited to, the human immunodeficiencyvirus (HIV) rev response element (RRE) (see e.g., Cullen et al. (1991)J. Virol. 65: 1053; and Cullen et al. (1991) Cell 58: 423), and thehepatitis B virus post-transriptional regulatory element (PRE) (seee.g., Huang et al. (1995) Molec. and Cell. Biol. 15(7): 3864; Huang etal. (1994) J. Virol. 68(5): 3193; Huang et al. (1993) Molec. and Cell.Biol. 13(12): 7476), and U.S. Pat. No. 5,744,326). Generally, the RNAexport element is placed within the 3′ UTR of a gene, and can beinserted as one or multiple copies. RNA export elements can be insertedinto any or all of the separate vectors generating the packaging celllines of the present invention.

The phrase “retroviral packaging cell line” refers to a cell line(typically a mammalian cell line) which contains the necessary codingsequences to produce viral particles which lack the ability to packageRNA and produce replication-competent helper-virus. When the packagingfunction is provided within the cell line (e.g., in trans by way of aplasmid vector), the packaging cell line produces recombinantretrovirus, thereby becoming a “retroviral producer cell line.”

The term “nucleic acid cassette” as used herein refers to geneticsequences within the vector which can express a RNA, and subsequently aprotein. The nucleic acid cassette contains the gene of interest. Thenucleic acid cassette is positionally and sequentially oriented withinthe vector such that the nucleic acid in the cassette can be transcribedinto RNA, and when necessary, translated into a protein or apolypeptide, undergo appropriate post-translational modificationsrequired for activity in the transformed cell, and be translocated tothe appropriate compartment for biological activity by targeting toappropriate intracellular compartments or secretion into extracellularcompartments. Preferably, the cassette has its 3′ and 5′ ends adaptedfor ready insertion into a vector, e.g., it has restriction endonucleasesites at each end. In a preferred embodiment of the invention, thenucleic acid cassette contains the sequence of a therapeutic gene usedto treat a hemoglobinopathic condition. The cassette can be removed andinserted into a vector or plasmid as a single unit.

As used herein, the term “gene of interest” refers to the gene insertedinto the polylinker of an expression vector. In one embodiment, the geneof interest encodes a gene which provides a therapeutic function for thetreatment of a hemoglobinopathy.

The term “promoter” as used herein refers to a recognition site of a DNAstrand to which the RNA polymerase binds. The promoter forms aninitiation complex with

RNA polymerase to initiate and drive transcriptional activity. Thecomplex can be modified by activating sequences termed “enhancers” orinhibitory sequences termed “silencers”.

As used herein, the term “cis” is used in reference to the presence ofgenes on the same chromosome. The term “cis-acting” is used in referenceto the controlling effect of a regulatory gene on a gene present on thesame chromosome. For example, promoters, which affect the synthesis ofdownstream mRNA are cis-acting control elements.

The term “suicide gene” is used herein to define any gene that expressesa product that is fatal to the cell expressing the suicide gene. In oneembodiment, the suicide gene is cis-acting in relation to the gene ofinterest on the vector of the invention, Examples of suicide genes areknown in the art, including HSV thymidine kinase (HSV-Tk).

The term “operably linked”, refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. In one embodiment, the term refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

The terms “transformation,” “transfection,” and “transduction” refer tointroduction of a nucleic acid, e.g., a viral vector, into a recipientcell.

The terms “pseudotype” or “pseudotyping” as used herein, refer to avirus whose viral envelope proteins have been substituted with those ofanother virus possessing preferable characteristics. For example, HIVcan be pseudotyped with vesicular stomatitis virus G-protein (VSV-G)envelope proteins, which allows HIV to infect a wider range of cellsbecause HIV envelope proteins (encoded by the env gene) normally targetthe virus to CD4 + presenting cells. In a preferred embodiment of theinvention, lentiviral envelope proteins are pseudotyped with VSV-G.

As used herein, the term “packaging” refers to the process ofsequestering (or packaging) a viral genome inside a protein capsid,whereby a virion particle is formed. This process is also known asencapsidation. As used herein, the term “packaging signal” or “packagingsequence” refers to sequences located within the retroviral genome whichare required for insertion of the viral RNA into the viral capsid orparticle. Several retroviral vectors use the minimal packaging signal(also referred to as the psi [Ψ] sequence) needed for encapsidation ofthe viral genome. Thus, as used herein, the terms “packaging sequence,”“packaging signal,” “psi” and the symbol “Ψ,” are used in reference tothe non-coding sequence required for encapsidation of retroviral RNAstrands during viral particle formation.

As used herein, the term “packaging cell lines” is used in reference tocell lines that do not contain a packaging signal, but do stably ortransiently express viral structural proteins and replication enzymes(e.g., gag, pol and env) which are necessary for the correct packagingof viral particles.

As used herein, the term “replication-defective” refers to virus that isnot capable of complete, effective replication such that infectivevirions are not produced (e.g. replication-defective lentiviralprogeny). The term “replication-competent” refers to wild-type virus ormutant virus that is capable of replication, such that viral replicationof the virus is capable of producing infective virions (e.g.,replication-competent lentiviral progeny).

As used herein, the term “incorporate” refers to uptake or transfer of avector (e.g., DNA or RNA) into a cell such that the vector can express atherapeutic gene product within the cell. Incorporation may involve, butdoes not require, integration of the DNA expression vector or episomalreplication of the DNA expression vector. As used herein, the term“erythroid-specific expression” or “red blood cell-specific expression”refers to gene expression which only occurs in erythocytes or red bloodcells (RBCs), used interchangeably herein.

The term “gene delivery” or “gene transfer” refers to methods or systemsfor reliably inserting foreign DNA into target cells, such as intomuscle cells. Such methods can result in transient or long termexpression of genes. Gene transfer provides a unique approach for thetreatment of acquired and inherited diseases. A number of systems havebeen developed for gene transfer into mammalian cells. See, e.g., U.S.Pat. No. 5,399,346. The lentiviral vector of the invention is optimizedto express antisickling proteins at therapeutic levels in virtually allcirculating RBCs.

The term “stem cell” refers to the cell from which a progenitor cell isderived. Stem cells are defined by their ability to self-renew. Stemcells include, for example, embryonic stem cells and somatic stem cells.Hematopoietic stem cells can generate daughter cells of any of thehematopoietic lineages. Stem cells with long term hematopoieticreconstituting ability can be distinguished by a number of physical andbiological properties from differentiated cells and progenitor cells(see, e.g., Hodgson, G. S. & Bradley, T. R., Nature, Vol. 281, pp381-382; Visser et al., J. Exp. Med., Vol. 59, pp. 1576-1590, 1984;Spangrude et al., Science, Vol. 241, pp. 58-62, 1988; Szilvassy et al.,Blood, Vol. 74, pp. 930-939, 1989; Ploemacher, R. E. & Brons, R. H. C.,Exp. Hematol., Vol. 17, pp. 263-266, 1989).

The term “embryonic stem cell” is used herein to mean anundifferentiated, pluripotent cell derived from a blastula stage embryo.

The term “somatic stem cell” is used here to mean cells in the bodywhich have the unique ability to regenerate themselves and differentiateinto many different types of cells. Examples of somatic stem cellsinclude blood stem cells, muscle/bone stem cells, brain stem cells, andliver stem cells.

As used herein, the term “progenitor” or“progenitor cells” refers tocells which are the precursors of differentiating cells. Many progenitorcells differentiate along a single lineage, but may have quite extensiveproliferative capacity. Examples of progenitor cells include, but arenot limited to, pluripotent stem cells, totipotent stem cells, myeloidstem cells, and lymphoid stem cells.

The term “globin” is used here to mean all proteins or protein subunitsthat are capable of covalently or noncovalently binding a heme moiety,and can therefore transport or store oxygen. Subunits of vertebrate andinvertebrate hemoglobins, vertebrate and invertebrate myoglobins ormutants thereof are included by the term globin. Examples of globinsinclude α-globin or variant thereof, β-globin or variant thereof, aγ-globin or a variant thereof, and δ-globin.

As used herein, “hematopoiesis,” refers to the formation and developmentof blood cells from progenitor cells as well as formation of progenitorcells from stem cells. Blood cells include but are not limited toerythrocytes or red blood cells (RBCs), reticulocytes, monocytes,neutrophils, megakaryotes, eosinophils, basophils, B-cells, macrophages,granulocytes, mast cells, thrombocytes, and leukocytes.

As used herein, the term “hemoglobinopathy” or “hemoglobinopathiccondition” includes any disorder involving the presence of an abnormalhemoglobin molecule in the blood. Examples of hemoglobinopathiesincluded, but are not limited to, hemoglobin C disease, hemoglobinsickle cell disease (SCD), sickle cell anemia, and thalassemias. Alsoincluded are hemoglobinopathies in which a combination of abnormalhemoglobins are present in the blood (e.g., sickle cell/Hb-C disease).

The term “sickle cell anemia” or “sickle cell disease” is defined hereinto include any symptomatic anemic condition which results from sicklingof red blood cells. Manifestations of sickle cell disease include:anemia; pain; and/or organ dysfunction, such as renal failure,retinopathy, acute-chest syndrome, ischemia, priapism and stroke.

As used herein the term “sickle cell disease” refers to a variety ofclinical problems attendant upon sickle cell anemia, especially in thosesubjects who are homozygotes for the sickle cell substitution in HbS.Among the constitutional manifestations referred to herein by use of theterm of sickle cell disease are delay of growth and development, anincreased tendency to develop serious infections, particularly due topneumococcus, marked impairment of splenic function, preventingeffective clearance of circulating bacteria, with recurrent infarcts andeventual destruction of splenic tissue. Also included in the term“sickle cell disease” are acute episodes of musculoskeletal pain, whichaffect primarily the lumbar spine, abdomen, and femoral shaft, and whichare similar in mechanism and in severity to the bends. In adults, suchattacks commonly manifest as mild or moderate bouts of short durationevery few weeks or months interspersed with agonizing attacks lasting 5to 7 days that strike on average about once a year. Among events knownto trigger such crises are acidosis, hypoxia and dehydration, all ofwhich potentiate intracellular polymerization of HbS (J. H. Jandl,Blood: Textbook of Hematology, 2nd Ed., Little, Brown and Company,Boston, 1996, pages 544-545). As used herein, the term “thalassemia”encompasses hereditary anemias that occur due to mutations affecting thesynthesis of hemoglobin. Thus, the term includes any symptomatic anemiaresulting from thalassemic conditions such as severe or β thalassemia,thalassemia major, thalassemia intermedia, α-thalassemias such ashemoglobin H disease.

As used herein, “thalassemia” refers to a hereditary disordercharacterized by defective production of hemoglobin. Examples ofthalassemias include β and α thalassemia. β thalassemias are caused by amutation in the beta globin chain, and can occur in a major or minorform. In the major form of β thalassemia, children are normal at birth,but develop anemia during the first year of life. The mild form of βthalassemia produces small red blood cells. α thalassemias are caused bydeletion of a gene or genes from the globin chain.

As used herein, “antisickling proteins” include proteins which preventor reverse the pathological events leading to sickling of erythrocytesin sickle cell conditions. In one embodiment of the invention, thelentiviral vector of the invention is used to deliver antisicklingproteins to a subject with a hemoglobinopathic condition. Antisicklingproteins also include mutated β-globin genes comprising antisicklingamino acid residues.

As used herein, the term “self-inactivating” or “SIN,” usedinterchangeably herein, refers to a vector which is modified, whereinthe modification greatly reduces the ability of the vector to mobilizeonce it has integrated into the genome of the recipient, therebyincreasing the safety of the use of the vector as a gene deliveryvector.

As used herein, the term “insulator” or “insulator element,” usedinterchangeably herein, refers to an exogenous DNA sequence that can beadded to a vector of the invention to prevent, upon integration of thevector into a host genome, nearby genomic sequences from influencingexpression of the integrated trans-gene(s). Conversely, the insulatorelement prevents the integrated vector from influencing expression ofnearby genomic sequences. This is generally achieved as the insulator isduplicated upon integration of the vector into the genome, such that theinsulator flanks the integrated vector (e.g., within the LTR region) andacts to “insulate” the integrated DNA sequence. Suitable insulators foruse in the invention include, but are not limited to, the chickenβ-Globin insulator (see Chung et al. Cell (1993) 74:505; Chung et al.,PNAS (1997) 94:575; and Bell et al. Cell 1999 98:387, incorporated byreference herein). Examples of insulator elements include, but are notlimited to, an insulator from an a-globin locus, such as chicken HS4.

II. Retroviral And Lentiviral Vectors

The present invention provides improved methods and compositions fortreating hemoglobinopathic conditions using retrovirus-based, e.g.,lentivirus-based, gene delivery vectors that hich achieve sustained,high-level expression of transferred therapeutic genes in eythroid cellsor erythroid precursor cells. In one embodiment of the invention, thevector comprises a self inactivating SIN vector. Particular lentiviralvectors of the invention are described by Pawliuk et al. (2001) Science294:2368 and Imren et al. (2002) PNAS 99:14380, incorporated byreference herein.

Retroviral and lentiviral vectors of the invention include, but are notlimited to, human immunodeficiency virus (e.g., HIV-1, HIV-2), felineimmunodeficiency virus (FIV), simian immunodeficiency virus (SIV),bovine immunodeficiency virus (BIV), and equine infectious anemia virus(EIAV). These vectors can be constructed and engineered usingart-recognized techniques to increase their safety for use in therapyand to include suitable expression elements and therapeutic genes, suchas those described below, which encode therapeutic proteins for treatingconditions including, but not limited to, hemoglobinopathies. In oneembodiment of the invention, the lentiviral vector is based on HIV-1.

In consideration of the potential toxicity of lentiviruses, the vectorscan be designed in different ways to increase their safety in genetherapy applications. For example, the vector can be made safer byseparating the necessary lentiviral genes (e.g., gag and pol) ontoseparate vectors as described, for example, in U.S. patent applicationSer. No. 09/311,684, the contents of which are incorporated by referenceherein. Thus, recombinant retrovirus can be constructed in which part ofthe retroviral coding sequence (gag, pol, env) is replaced by a gene ofinterest rendering the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions through the use of ahelper virus or a packaging cell line, by standard techniques. Protocolsfor producing recombinant retroviruses and for infecting cells in vitroor in vivo with such viruses can be found in Current Protocols inMolecular Biology, Ausubel, F.M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals.

A major prerequisite for the use of viruses as gene delivery vectors isto ensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment packaging cell lines, which produce onlyreplication-defective retroviruses, has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). Accordingly, in oneembodiment of the invention, packaging cell lines are used to propagatevectors (e.g., lentiviral vectors) of the invention to increase thetiter of the vector virus. The use of packaging cell lines is alsoconsidered a safe way to propagate the virus, as use of the systemreduces the likelihood that recombination will occur to generatewild-type virus. In addition, to reduce toxicity to cells that caused byexpression of packaging proteins, packaging systems can be use in whichthe plasmids encoding the packaging functions of the virus are onlytransiently transfected by, for example, chemical means.

In another embodiment, the vector can be made safer by replacing certainlentiviral sequences with non-lentiviral sequences. Thus, lentiviralvectors of the present invention may contain partial (e.g., split) genelentiviral sequences and/or non-lentiviral sequences (e.g., sequencesfrom other retroviruses) as long as its function (e.g., viral titer,infectivity, integration and ability to confer high levels and durationof therapeutic gene expression) are not substantially reduced. Elementswhich may be cloned into the viral vector include, but are not limitedto, promoter, packaging signal, LTR(s), polypurine tracts, RRE, etc.

In one embodiment, the retroviral vector of the invention comprises aleft (5′) retroviral LTR; a retroviral export element, optionally alentiviral reverse response element (RRE); a promoter, or active portionthereof, and a locus control region (LCR), or active portion thereof,operably linked to a gene of interest; and a right (3′) retroviral LTR.Retroviral vectors, including lentiviral vectors, of the invention canfurther contain a central polypurine tract (cPPT) or DNA flap. ThecPPT/DNA flap is used to increase viral titers and transductionefficiency. In a particular embodiment, the cPPT/DNA flap is from HIV-1.In another embodiment, the cPPT/DNA flap increases the efficiency oftransfection in to HSCs.

In another embodiment of the invention, the LTR region is modified byreplacing the viral LTR promoter with a heterologous promoter. In oneembodiment, the promoter of the 5′ LTR is replaced with a heterologouspromoter. Examples of heterologous promoters which can be used include,but are not limited to, the cytomegalovirus (CMV) promoter which iseffective for high level expression.

Retroviral vectors of the invention also include vectors which have beenmodified to improve upon safety in the use of the vectors as genedelivery agents in gene therapy. In one embodiment of the invention, anLTR region, such as the 3′ LTR, of the vector is modified in the U3and/or U5 regions, wherein a SIN vector is created. Such modificationscontribute to an increase in the safety of the vector for gene deliverypurposes. In one embodiment, the SIN vector of the invention comprises adeletion in the 3′ LTR wherein a portion of the U3 region is replacedwith an insulator element. The insulator prevents the enhancer/promotersequences within the vector from influencing the expression of genes inthe nearby genome, and vice/versa, to prevent the nearby genomicsequences from influencing the expression of the genes within thevector. In a further embodiment of the invention, the 3′ LTR is modifiedsuch that the U5 region is replaced, for example, with an ideal poly(A)sequence. It should be noted that modifications to the LTRs such asmodifications to the 3′ LTR, the 5′ LTR, or both 3′ and 5′ LTRs, arealso included in the invention.

Retroviral vectors of the invention can also comprise elements whichcontrol selection of the transduced cell. In one embodiment, theretroviral vector comprises a nucleic acid cassette which allows for invivo selection of the transduced cell. For example, the nucleic acidcassette could contain the cDNAs for methylguanine methyltransferase(MGMT) or the human glutathione-S-transferase pi (GST pi) which haveboth been successfully used as in vivo selection markers for transducedhematopoietic stem cells. These transgenes provide chemoprotection to tothe combination of O6-benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or to transduced cells againstposttransplant treatment with cyclophosphamide, respectively. In anotherembodiment of the invention, the retroviral vector contains a suicidegene operably linked to a promoter. Examples of suicide genes include,but are not limited to, herpes simplex virus (HSV) thymidine kinase(HSV-Tk).

The infectivity of retroviruses, including lentiviruses, is dependentupon the interaction between glycoproteins displayed on the surface ofthe viral particle and receptors found on the surface of the targetcell. HIV is only able to infect T-cells that display the CD4+receptoron their cell surfaces. To maximize the infectivity of an HW-based genedelivery system, the lentivirus can be pseudotyped to display aglycoprotein known to bind a wider range of cell type than HIV. In oneembodiment of the invention, the recombinant lentivirus is pseudotypedwith the vesicular stomatitis virus G coat protein (VSV-G). Pseudotypingwith VSV-G increases both the host range and the physical stability ofthe viral particles, and allows their concentration to very high titersby ultracentrifugation (Naldini et al. (1996), supra; Aiken (1997) J.Virol. 71:5871-5877; Akkina et al., supra; Reiser et al. (1996) Proc.Natl. Acad. Sci. USA 93:15266-15271). In a preferred embodiment, thelentiviral vector of the invention is transduced into hematopoietic stemcells (HSCs) after pseudotyping with VSV-G and concentration.

The promoter of the lentiviral vector can be one which is naturally(i.e., as it occurs with a cell in vivo) or non-naturally associatedwith the 5′ flanking region of a particular gene. Promoters can bederived from eukaryotic genomes, viral genomes, or synthetic sequences.Promoters can be selected to be non-specific (active in all tissues),tissue specific, regulated by natural regulatory processes, regulated byexogenously applied drugs, or regulated by specific physiological statessuch as those promoters which are activated during an acute phaseresponse or those which are activated only in replicating cells.Non-limiting examples of promoters in the present invention include theretroviral LTR promoter, cytomegalovirus immediate early promoter, SV40promoter, dihydrofolate reductase promoter, and cytomegalovirus (CMV).The promoter can also be selected from those shown to specificallyexpress in the select cell types which may be found associated withconditions including, but not limited to, hemoglobinopathies. In oneembodiment of the invention, the promoter is cell specific such thatgene expression is restricted to red blood cells. Erythrocyte-specificexpression is achieved by using the human β-globin promoter region andlocus control region (LCR).

Retroviral vectors, including lentiviral vectors, of the inventionoptionally can also contain one or more elements that allow for thecorrect expression of the nucleic acid cassette, i.e. therapeutic geneof interest. In one embodiment of the invention, the gene of interest isa gene which is used to treat or reduce the detrimental effects of ahemoglobinopathic condition. Such genes include those encodingantisickling proteins that can be used to treat a hemoglobinopathy,wherein the antisickling protein is used to prevent or reverse thepathological events leading to sickling of erythrocytes in sickle cellconditions. For example, a globin gene, such as, β-globin, 6-globin, orα-globin gene, can be expressed using the retroviral vectors of theinvention to treat hemoglobinopathies via gene therapy. In oneembodiment, human β-globin is used to treat a subject who has ahemoglobinopathy, such as sickle cell disease or thalassemia.

Suitable β-globin genes for use in the present invention includewild-type and variant genes. In one embodiment, the β-globin gene ishuman β^(A)-globin gene. Variant β-globin genes include those geneswhich contain additions/deletions and mutant versions of the gene whichhave altered characteristics, including improved antisicklingproperties. In one embodiment, the β-globin gene comprises one or moredeletions of intron sequences. In another embodiment, the β-globin is amutant human β-globin gene encoding at least one antisickling aminoacid. Antisickling amino acids can be identified using standardalignment programs aligning β-globin with δ-globin and/or α-globin, thusderiving the antisickling amino acids from the δ-globin and/or α-globinprotein sequences. In other embodiments, the human β-globin gene is thehuman β^(A)-globin gene encoding a threonine to glutamine mutation atcodon 87 (β^(A-T87Q)).

One skilled in the art will recognize that the selection of the promoterto express the gene of interest will depend on the vector, the nucleicacid cassette, the cell type to be targeted, and the desired biologicaleffect. One skilled in the art will also recognize that in the selectionof a promoter the parameters can include: achieving sufficiently highlevels of gene expression to achieve a physiological effect; maintaininga critical level of gene expression; achieving temporal regulation ofgene expression; achieving cell type specific expression; achievingpharmacological, endocrine, paracrine, or autocrine regulation of geneexpression; and preventing inappropriate or undesirable levels ofexpression. Any given set of selection requirements will depend on theconditions but can be readily determined once the specific requirementsare determined. In one embodiment of the invention, the promoter is cellspecific such that gene expression is restricted to red blood cells.Erythrocyte-specific expression is achieved by using the human β-globinpromoter region and locus control region (LCR).

Standard techniques for the construction of expression vectors suitablefor use in the present invention are well-known to those of ordinaryskill in the art and can be found in such publications as Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold SpringHarbor, N.Y. A variety of strategies are available for ligatingfragments of DNA, the choice of which depends on the nature of thetermini of the DNA fragments and which choices can be readily made bythe skilled artisan.

Gene therapy vectors of the present invention, such as the foregoingretroviral vectors, including lentiviral vectors, can be used to expressa variety of therapeutic proteins in transformed erythroid cells. In oneembodiment, the gene of interest to be expressed in the vector is a genewhich can be used to treat a hemoglobinopathy, such as the humanβ-globin gene or a variant thereof. Variants of human β-globin aredescribed in the examples below, and include, for example, β-globinvariants which include a substitution of threonine at position 87 withglutamine [β^(A87) Thr:Gln (β^(A-T87Q))]. The gene of interest can beobtained for insertion into the viral vector through a variety oftechniques known to one of ordinary skill in the art.

Particular gene therapy vectors of the invention include, but are notlimited to, the lentiviral vectors shown in FIG. 1 c, FIG. 13, and FIG.14. This HIV-based recombinant lentiviral vector contains, in a 5′ to 3′direction, the 5′ flanking HIV LTR, a packaging signal or Ψ+, a centralpolypurine tract or DNA flap of HIV-1 (cPPT/FLAP), a Rev-responseelement (RRE), the human β-globin gene 3′ enhancer, a gene of interest,such as the human β-globin gene variant containing the β^(A87) Thr:Glnmutation, 266 by of the human β-globin promoter, 2.7 kb of the humanβ-globin LCR, a polypurine tract (PPT), and the 3′ flanking HIV LTR. TheLTR regions further comprise a U3 and U5 region, as well as an R region.The U3 and U5 regions can be modified together or independently tocreate a vector which is self-inactivating, thus increasing the safetyof the vector for use in gene delivery. The U3 and U5 regions canfurther be modified to comprise an insulator element. In one embodimentof the invention, the insulator element is chicken HS4. cDNA of thetherapeutic gene of interest, such as, for example, human β-globin, isamplified by PCR from an appropriate library. The gene is cloned into aplasmid, such as pBluescript II KS (+) (Stratagene), containing adesired promoter or gene-expression controlling elements, such as thehuman β-globin promoter and LCR elements. Following restriction enzymedigestion, or other method known by one skilled in the art to obtain adesired DNA sequence, the nucleic acid cassette containing the promoterand LCR elements and therapeutic gene of interest is then inserted intoan appropriate cloning site of the lentiviral vector, as shown in FIG. 1c.

The step of facilitating the production of infectious viral particles inthe cells may be carried out using conventional techniques, such asstandard cell culture growth techniques. If desired by the skilledartisan, lentiviral stock solutions may be prepared using the vectorsand methods of the present invention. Methods of preparing viral stocksolutions are known in the art and are illustrated by, e.g., Y. Soneokaet al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al.(1992) J. Virol. 66:5110-5113. In a method of producing a stock solutionin the present invention, lentiviral-permissive cells (referred toherein as producer cells) are transfected with the vector system of thepresent invention. The cells are then grown under suitable cell cultureconditions, and the lentiviral particles collected from either the cellsthemselves or from the cell media as described above. Suitable producercell lines include, but are not limited to, the human embryonic kidneycell line 293, the equine dermis cell line NBL-6, and the canine fetalthymus cell line Cf2TH.

The step of collecting the infectious virus particles also can becarried out using conventional techniques. For example, the infectiousparticles can be collected by cell lysis, or collection of thesupernatant of the cell culture, as is known in the art. Optionally, thecollected virus particles may be purified if desired. Suitablepurification techniques are well known to those skilled in the art.

Other methods relating to the use of viral vectors in gene therapy canbe found in, e.g., Kay, M. A. (1997) Chest 111 (6 Supp.):1385-1425;Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory,Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin.Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther.7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M.(1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994)Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opin.Investig. Drugs 8:2159-2172; Smith-Arica, J. R. and Bartlett, J. S.(2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C. et al. (2000) Nature408:483-8.

Retroviral vectors, including lentiviral vectors, as described above canbe administered in vivo to subjects by any suitable route, as is wellknown in the art. The term “administration” refers to the route ofintroduction of a formulated vector into the body. For example,administration may be intravenous, intramuscular, topical, oral, or bygene gun or hypospray instrumentation. Thus, administration can bedirect to a target tissue or through systemic delivery. Administrationdirectly to the target tissue can involve needle injection, hypospray,electroporation, or the gene gun. See, e.g., WO 93/18759, herebyincorporated by reference herein.

Alternatively, the retroviral vectors of the invention can beadministered ex vivo or in vitro to cells or tissues using standardtransfection techniques well known in the art.

The retroviral vectors of the invention can also be transduced into hostcells, including embryonic stem cells, somatic stem cells, or progenitorcells. Examples of progenitor host cells which can be transduced by theretroviral vectors of the invention include precursors of erythrocytesand hematopoietic stem cells. In another embodiment, the host cell is anerythrocyte. Transduced host cells can be used as a method of achievingerythroid-specific expression of the gene of interest in the treatmentof hemoglobinopathies.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Preferably, the carrier is suitable foradministration directly into an affected joint. The carrier can besuitable for intravenous, intraperitoneal or intramuscularadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Another aspect of the invention pertains to pharmaceutical compositionsof the lentiviral vectors of the invention. In one embodiment, thecomposition includes a lentiviral vector in a therapeutically effectiveamount sufficient to treat or prevent (e.g. ameliorate the symptoms of ahemoglobinopathy), and a pharmaceutically acceptable carrier. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, such as treatment or prevention of ahemoglobinopathic condition. A therapeutically effective amount oflentiviral vector may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thelentiviral vector to elicit a desired response in the individual. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the lentiviral vector are outweighed by thetherapeutically beneficial effects. The potential toxicity of thelentiviral vectors of the invention can be assayed using cell-basedassays or art recognized animal models and a therapeutically effectivemodulator can be selected which does not exhibit significant toxicity.In a preferred embodiment, a therapeutically effective amount of alentiviral vector is sufficient to treat a hemoglobinopathy.

Sterile injectable solutions can be prepared by incorporating lentiviralvector in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens can be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The amount of viral vector in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

A major advantage of retroviral vectors, including lentiviral vectors,is that they are capable of integrating into the genome of a host celland, therefore, enable long term expression of therapeutic proteins.Lentiviral vectors have been successfully used to deliver exogenousgenes both in vitro and in vivo to a large variety of cell populationsin several species, including neurons of the central nervous system(Naldini et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388),retinal cells (Miyoshi et al. (1997) Proc. Natl. Acad. Sci. USA94:10319-10323), and pancreatic cells (Giannokakis et al. (1999) GeneTher. 6:1545-1551).

The invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Materials And Methods Whole-Blood p50 Analysis

In the SCD mouse model experiments, p50 measurements on whole blood wereperformed using a Hemoscan (Aminco, Silver Spring, Md.). Theconcentration of deoxygenated HbS in equilibrium with the polymer (CSAT)was determined using the p50 method (R. E. Benesch et al. (1978)Analytical Biochem. 89:162) for various mixtures of HbS with testhemoglobins (1:1 ratio). p50 is the oxygen pressure at which half of themolecules of Hb are deoxygenated, as detected spectroscopically. Becausethe HbS polymer has a much lower oxygen affinity than HbS in solution, asudden jump in p50 indicates the start of the formation of the polymer,and the concentration of Hb at which the jump occurs gives the CSATvalue, expressed in g/dl

Vector Construction And Virus Production

The human beta-globin gene including its promoter and 3′ enhancersequence was cloned from BGT9 (Pasceri, et al. (1998) Blood 92:653). A374bp fragment was deleted from intron 2 and hypersensitive sites 2-4were cloned from BGT33 (Rubin, et al. (2000) Blood 95:3242). Virusstocks were generated by transient transfection of 293T cells with therecombinant lentiviral vector together with separate plasmids expressingHIV-1 Gag-Pol, Rev and Tat. DNA sequence of the vector will be providedupon request. One liter of virus was concentrated by ultracentrifugationat 25,000 RPM for 90 minutes at 4° C. and the viral pellet resuspendedin 300 μl of serum free medium (Life Technologies, Frederick, Md.). Theabsence of replication competent retrovirus (RCR) was verified bymobilization assay as described (Pawliuk, et al. (1994) Blood 84:2868).Viral titers were determined by Southern blot analysis.

RNA And DNA Analyses

In the SCD mouse model experiments, Southern blot analysis was performedusing standard methods (Pawliuk et al., supra). A ³²P-labeled exonicfragment of the human β-globin gene was used as a probe. Quantificationof vector copy number was achieved by densitometry using a phosphoimagerwith ImageQua™ software (Molecular Dynamics, Sunnyvale, Calif.). DNA wasdigested with Afl II and probed with an exonic fragment of the humanβ-globin gene.

In the THAL experiments, human β globin RNA in peripheral reticulocyteswas quantified by RNase protection assay as described in Leboulch et al.(1994) EMBO 13:3065. A 1.6-kb BamH1 fragment of the human β globin gene,[³²P]dCTP-labeled by random priming, was used as a probe.

THAL Mouse Model

β-Thalassemia mice homozygous for a deletion of the murine β-major gene(C57BL/6 Hbb^(th-1)/Hbb^(th-1)) (Skow et al. (1983) Cell 34:1043)hereafter referred to as THAL mice, and control C57BL/6(B6) mice werebred from parental stocks obtained from The Jackson Laboratory. Theidentity of homozygous THAL mice was confirmed by isoelectric focusinganalysis of RBC lysates to detect characteristic single, slow-migratingHb tetramers consisting of two murine α and two murine β minor globinchains.

Bone Marrow Transduction And Transplantation

In the SCD mouse model experiments, donor mice were injected 4 daysbefore bone marrow harvest with 150 mg/kg of 5-fluorouracil (5-FU).Cells were prestimulated overnight in serum free medium (LifeTechnologies, Frederick, Md.) supplemented with 200 mM L-glutamine, 6ng/ml of murine Interleukin-3, 10 ng/ml of human Interleukin-6, 10 ng/mlof murine Interleukin-1α and 100 ng/ml of murine Stem Cell Factor(Peprotech, Rocky Hill, N.J.). Cells were exposed to concentrated viralsupernatants on Retronectin™ (Biowhittaker, East Rutherford, N.J.)coated plates for 5-6 hours in the presence of 8 μg/ml of protaminesulfate (Sigma, St. Louis, Mo.). Following infection, cells wereharvested and injected, without selection, into recipient mice given1100 cGy (¹²³Cs γ-rays) of total body irradiation (split dose of 550 cGyover 3 hours).

For experiments using THAL mice, bone marrow cells (24×10⁶) from maleTHAL mice injected intravenously 4 days previously with 5-fluorouracil(100 mg/kg) were stimulated overnight in Iscove's medium supplementedwith 1% BSA, 10 μg/ml bovine pancreatic insulin, and 200 μg/ml humantransferrin (BIT; StemCell Technologies, Vancouver), 10⁻⁴ M2-mercaptoethanol, 2 mM glutamine, 10 ng/ml human interleukin-11 (IL-11,Genetics Institute, Cambridge, Mass.), 100 ng/ml human flt3-ligand(Immunex, Seattle, Wash) and 300 ng/ml murine steel factor (expressed inCOS cells and purified at the Terry Fox Laboratories, Vancouver). Thenext day, harvested cells were pelleted and resuspended in 0.9 ml of theaforementioned medium containing the same growth factor combination withconcentrated, vesicular stomatitis virus glycoprotein-G-pseudotyped GFP-or β globin-lentivirus at a final virus concentration of 1.5×10⁹infectious units/ml (functional titer measured by Southern blot analysisof transduced NIH 3T3 cells). Infection was performed for 5 h onfibronectin (5 μg/cm², Sigma)-coated Petri dishes in the presence of 5μg/ml protamine sulfate. After infection, 2×10⁶ cells were transplanted,without selection, by i.v. injection into each female THAL recipientgiven 900 cGy (110 cGy/min 37 Cs γ-rays) of total body irradiation.

FACS Analysis

RBCs were washed in PBS, fixed, permeabilized and stained with anFITC-labeled monoclonal antibody that specifically recognizes human HbA(PerkinElmer Wallac, Norton, Ohio). Samples were analyzed on a FACScanflow cytometer (Becton Dickinson, San Diego, Calif.).

Primer Extension Analysis

In the SCD mouse model experiments, total RNA was extracted from100-2001 of blood using TRIzol reagent (LifeTechnologies, Frederick,Md.). Primer extension was performed using the Primer ExtensionSystem-AMV Reverse Transcriptase kit (Promega, Madison, Wis.) accordingto the manufacturer's instructions. Primers for human β^(A)-globin[5′-CAGTAACGGCAG ACTTCTCCTC-3′] (SEQ ID NO:1) and mouse β^(single)-globin [5′-TGATGTCTGTTTC TGGGGTTGTG-3′] (SEQ ID N0:2) generate extensionproducts of 90 bp and 53 bp respectively. Extension products wereradioactively labeled by including ³²P dCTP in the reaction mixture.Reactions were performed using 1 μg of RNA and run on a denaturing 7%polyacrylamide gel. Radioactive bands were quantified by phosphoimageranalysis. Measurements were corrected for the number of dCTP residues inhuman (Rubin et al., supra) and mouse (Papadea et al., supra) extensionproducts.

Protein Analysis

In the SCD mouse model experiments, quantification of β-globin chainsand Hb by HPLC was performed as described in Fabry et al. (1995) Blood86:2419 and Papadea et al. (1996) Clin. Chem. 42:57.

RBC Sickling

In the SCD mouse model experiments, sickling of erythrocytes was studiedas a function of PO2, from 0 to 150 mm Hg. Blood was diluted with PBS(340 mOsm) containing 5 mM glucose and 0.5 g/dl of bovine serum albumin.Cells were equilibrated with a mixture of air and nitrogen at thedesired PO2, for 30 minutes at 37° C. in a rotary shaker, and fixed bythe addition of 4% formaldehyde equilibrated at the same PO2 andtemperature. The reversal of sickling was determined after incubation ofthe fully deoxygenated cell suspension by reoxygenation with air at 0°C. for one hour, prior to fixation with the formaldehyde solutionequilibrated with air at 0° C. Proportions of sickle and non-sicklecells were determined by microscopy using Nomarski optics.

Hematology

In the SCD mouse model experiments, red cell counts and total Hb weremeasured on a CBC analyzer (CBC Technologies, Oxford, Conn.).Reticulocytes were analyzed using the Sysmex SE 9000 system (Sysmex Corpof America, Long Grove, Ill.).

Urine Concentrating Ability

In the SCD mouse model experiments, mice were deprived of water for 24hours. At the end of this period, urine was collected onto Parafilm andthe osmolarity measured after a 1:10 dilution with distilled water usinga Microosmette (Precision Systems, Natick, Mass.).

RBC Density Gradients

In the SCD mouse model experiments, density gradients were performed aspreviously described in Fabry et al. (1991) Blood 78:217.

Delay Time of HbS Polymerization

In the SCD mouse model experiments, polymer formation upon deoxygenationof purified Hb or membrane free hemolysates was studied by measuring thedelay time of polymerization with the method described by Adachi andAsakura (Adachi, et al. (1980) J. Mol. Biol. 144:467). In brief, after atemperature jump from 0° C. to 30° C. of the deoxygenated samples in asolution of 1.80 M potassium phosphate (pH 7.4) and 2 mM of sodiumdithionite, the turbidity induced by HbS polymerization was recorded at700 nm The probability factor for nucleation was derived from themeasurement of the delay time performed at various Hb concentrations.

Globin Protein Analysis

In the THAL mouse experiments, the proportion of RBCs expressing human βglobin protein was assessed by fluorescence-activated cell sorter (FACS)analysis of RBCs that had been fixed and stained with a biotinylatedanti-human antibody (Perkin-Elmer) and Streptavidin-PE as described inKalberer et al. (2000) PNAS 97:5411. RBC lysates from freshly collectedblood were analyzed by isoelectric focusing by using the Resolve Hb testkit (Perkin-Elmer) as described in Fabry et al. (1992) PNAS 89:12155.The globin composition was determined by HPLC with a denaturing solventthat separates the globin chains and a Vydac large-pore (3,000 A) C₄column with a modified acetonitril/H₂O/trifluoroacetic acid gradient asdescribed in Fabry et al., supra. The amount of unpaired α globin chainsassociated with RBC membranes was determined by ureaTriton-polyacrylamide gel electrophoresis analysis as described inRouyer-Fessard et al. (1989) J. Biol. Chem. 264:19092. In brief, afterextensive washing of membrane ghosts, loading of equivalent amounts ofprotein, and Coomassie blue staining of the gel, proteins were analyzedby densitometry at 570 nm. The proportion of membrane-associated αglobin chain was expressed as a percentage of total membrane proteins.

Hematologic Parameters And RBC Density Gradient

In the THAL mouse experiments, blood from the tail vein was used toanalyze RBC indices and reticulocyte counts by using the Sysmex SE 9500system (Sysmex Corp. of America, Long Grove, Ill.). Blood smears werestained with methylene blue for manual reticulocyte counts to validatethe Sysmex reticulocyte counts in the majority of cases and thesenumbers correlated well. Blood smears were also stained withWright-Giemsa by using an automatic stainer. Smears were reviewedblinded by two independent hematologists. RBC densities were examined onPercoll-Larex gradients (Larex International, St Paul), as described inFabry et al. (1984) Blood 90:3332. The Student's t test was used todetermine whether hematological parameters differed between treatmentgroups.

Histopathology

In the THAL mouse experiments, livers and spleens were fixed in 10%neutral buffered formalin. Tissues were paraffin-embedded.Five-micrometer sections were stained with hematoxylin-eosin and Perlsiron stain and subsequently examined by light microscopy.

Example I Construction of Human β^(A)-Globin Gene Variant

In order to take advantage of the fact that γ-globin is a stronginhibitor of HbS polymerization, the human β^(A)-globin gene was mutatedin such a way as to emulate the antisickling activity of γ-globin. Ahuman β^(A)-globin gene variant was mutated at codon 87 to encode aGlutamine [β^(A87) Thr:Gln (β^(A-T87Q))], which is thought to beresponsible for most of the antisickling activity of γ-globin (Nagel etal. (1979) PNAS USA 767:670).

In order to study the antisickling capacity and oxygen-binding affinityof the human β^(A)-globin gene variant, transgenic mice were made whichexpressed both human β^(A-T87Q)-and α-globins, but neither mouse α- normouse β-globin. These mice had normal hematological parameters andviability, and the β^(A-T87Q)-globin variant extracted from the RBCs wasfound to be almost as potent an inhibitor of HbS polymerization asγ-globin in vitro and much more so than β^(A)-globin, as shown in FIG. 1a. FIG. a shows that HbA-T87Q and HbF are potent inhibitors of HbSpolymerization in vitro in contrast to HbA. As shown in FIG. 1 a, wholeblood analysis of p50, the pO₂ at which 50% of the Hb molecules areoxygenated, showed that the oxygen-binding affinity of β^(A-T87Q) Hb waswell within the range observed with wild-type β^(A) Hb in mice: 31.1±0.2mm Hg (standard error=SE) versus 32.7±1.8 mmHg (SE), respectively.

The β^(A-T87Q)-globin variant was then inserted in a lentiviral vectorwhich was optimized for transfer to HSCs and erythroid-specificexpression. The central polypurine tract/DNA flap of HIV-1 (Zennou etal. (2000) Cell 101:173) was incorporated in the construct to increaseviral titers and transduction of HSCs after pseudotyping with thevesicular stomatitis virus glycoprotein G (VSV-G) and concentration, asshown in Figure lc. Southern blot analysis (Figure lb) showed titersreaching 1.5×109 infectious units per ml after pseudotyping withVesicular Stomatitis Virus glycoprotein-G (VSV-G) and physicalconcentration. Specific LCR elements were chosen on the basis of resultsof single integrants in erythroid cells assessed by recombinase-mediatedcassette exchange (Bouhassira et al. (1997) Blood 90:3332).

Example II Analysis of β^(A-T87Q)-Globin Lentivirus

The β^(A-T87Q)-globin lentivirus was first analyzed in lethallyirradiated normal syngeneic C57BL/6 recipient mice in the absence of anyselection. Proviral transfer was stable with an average copy number of3.0±0.5 (SE) per genome of peripheral nucleated blood cells 3 monthsafter transplantation, as shown in FIG 1 b. At 10 months aftertransplantation, all mice expressing human β^(A-T87Q)-globin proteinwith up to 99% (mean 96±0.9% (SE)) of their RBCs staining positive withan antibody that specifically recognizes human β-globin in this case,the β^(A-T87Q) variant (FIG. 2 c). No β^(A-T87Q)-globin expression wasdetected in other blood lineages by antibody staining. Humanβ^(A-T87Q)-globin protein represented up to 22.5% [mean 16±3.1% (SE)] ofendogenous mouse β-chains in recipients of β^(A-T87Q)-globinlentivirus-transduced bone marrow, as determined by high-performanceliquid chromatography (HPLC) (FIG. 2 c). The fourfold discrepancybetween human β^(A-T87Q)-globin mRNA and protein levels is consistentwith differences observed in mice transgenic for the β^(A)-globin gene(Alami et al. (1999) Blood Cells Mol. Dis. 25:110).

Long term secondary transplants were also performed with bone marrowfrom a representative primary recipient killed 5 months aftertransplantation. Fluorescence-activated cell sorting (FACS) analysis ofperipheral blood samples of secondary recipients 4 months aftertransplantation showed that 87±2.3 (SE) of RBCs expressed high levels ofhuman β^(A-T87Q)-globin protein, thus demonstrating that transduction oftrue HSCs was achieved. Analysis of position effect variegationsuggested that pan-cellular expression was the result of balancedexpression from polyclonal stem cell reconstitution with multiplechromosomal integration sites rather than true position-independentexpression.

Example III Gene Therapy of Mouse Models Using Retroviral Vectors InVivo Analysis of β^(A-T87Q)-Globin Lentivirus

In order to study the efficacy of the β^(A-T87Q)-globin lentiviralvector in vivo, two different SCD transgenic mouse models were used: SAD(Trudel et al. (1991) EMBO J. 10:3157) and Berkeley (BERK) (Paszty et al(1997) Science 278:876). SAD mice express human α-globin together with a“super S” globin resulting from two point mutations added to the humanβ^(S) gene (Trudel et al., supra), whereas BERK mice, which expresshuman α and human β^(S)-globulins, do not express any murine globinsbecause of complete disruption of both mouse α and β-globin gene loci(Paszty et al., supra). The phenotype of BERK mice is overall moresevere than that of SAD mice, although some of the hematologicalabnormalities in BERK mice are caused by an associated β-thalassemicsyndrome due to suboptimal expression of the transgenic human β^(S) gene(Nagel et al. (2001) Br. J. Haematol. 112:19).

SAD and BERK bone marrow was transduced with the β^(A-T87Q)-globinlentiviral vector and transplanted into lethally irradiated syngeneicC57BL/6 mouse recipients. Transduced SAD marrow was also transplantedinto lethally irradiated syngeneic SAD recipients. Three months aftertransplantation, reconstitution of recipient C57BL mice with donor BERKor SAD bone marrow was essentially complete for all mice, as determinedby quantification of murine β-single Hb by HPLC (FIG. 3).

Isoelectric focusing electrophoresis of blood samples from mice 3 monthsafter transplantation shows all of the expected species of Hb, as shownin FIG. 4. The amount of β^(A-T87Q)-globin expressed in the transplantedmice, as measured by Hb HPLC, was up to 108% (mean 75.5±17.1% (SE)) and51% (mean 42.5±5.5% (SE)) of the transgenic HbS for recipients ofβ^(A-T87Q)-globin lentivirus-transduced BERK and SAD bone marrow,respectively, as shown in FIG. 3. These values correspond to up to 52%and 12% of the total Hb of BERK and SAD mice, respectively. The greateramount of β^(A-T87Q)-globin-containing Hb observed in erythrocytesderived from transduced bone marrow cells of BERK mice as compared toSAD mice may be explained by the absence of the murine β-globulin mRNAand the associated thalassemic phenotype of BERK mice, which favorstranslation of the added β^(A-T87Q)-globin mRNA species (Nagel et al,supra, 2001).

In Vivo Analysis of Polymerization Inhibition

In order to determine whether β^(A-T87Q)-globin was capable ofinhibiting HbS polymerization in vivo in transplanted SCD mouse models,the morphology of RBCs from transplanted mice was analyzed as a functionof oxygen pressure in vitro. Examination of the obtained sigmoidsickling curves showed a marked change in the proportion of sickledcells, as shown in FIGS. 5 a and 5 b. For recipients ofβ^(A-T87Q)-globin lentivirus-transduced BERK marrow, the greatestdifference occurred at 5% pO₂, with 80±1.7% (SE) versus 26±7.5% (SE)(P=0.01) sickle cells for mock-transduced and β^(A-T87Q)-globinlentivirus-transduced marrow, respectively. In comparison, analysis ofRBCs from humans with sickle trait, who are heterozygous for the β^(S s)allele and asymptomatic, showed ˜40% sickled cells at 5% pO₂, with 81±3%(SE) versus 46±11% (SE) (P=0.03) sickle cells for mock-transduced andβ^(A-T87Q)-globin lentivirus-transduced marrow, respectively.Examination of peripheral blood smears at ambient pO₂ showed aneightfold decrease in the proportion of irreversibly sickled cells(ISCs) in mice transplanted with β^(A-T87Q)-globin lentivirus-transducedBERK marrow with complete disappearance of highly dehydrated ISCs. ForSAD mice, no ISCs could be detected after β^(A-T87Q)-globinlentivirus-transduction, as shown below in Table 1. Table 1 shows thecorrection of hematological abnormalities and urine concentrating defectin recipients of β^(A-T87Q)-globin-transduced BERK bone marrow.

TABLE 1 Correction of hematological abnormalities and urineconcentrating defect in recipients of β^(A-T87Q)-globin-transduced BERKbone marrow Urine concentrations RBCs Hb Reticulocytes ISCs † (mOsM)(number of Mice * (10⁶/μl) (g/dl) (%) (%) mice) C57BL/6 controls (n = 3)10.1 ± 0.3  15.0 ± 0.6 4.1 ± 0.6 —  3247 ± 500 (n = 22) BERK controls (n= 3) 7.4 ± 0.6  9.4 ± 0.9 17.8 ± 0.6  16.0 ‡  1452 ± 331 (n = 4) BERKβ^(A-T87Q) (n = 3)  10.1 ± 1.1 §   13.0 ± 0.41 ∥   5.8 ± 1.8 ¶ 2.0 #  3600 ± 381 (n = 2) ** SAD control (n = 4) 8.4 ± 0.6 13.0 ± 0.6 3.4 ±1.2 2.6 # 3840 ± 175 (n = 3) SAD β^(A-T87Q) (n = 3) 8.7 ± 0.1 13.7 ± 0.22.8 ± 0.1 0   3920 ± 326 (n = 3) RBCs, red blood cells; Hb, hemoglobin;ISCs, irreversibly sickled cells. Values shown with SE and statisticalsignificance established by Student's t test. * n is the number of micefor RBCs, Hb, and reticulocytes. † A total of 2000 RBCs were examinedfrom BERK control and BERK β^(A-T87Q) mice (n = 2) and 3000 RBCs wereexamined from SAD control and SAD β^(A-T87Q)mice (n = 2). § P = 0.15with substantial correction of anisocytosis and poikilocytosis. ∥ P =0.01. ¶ P = 0.05. # Only hydrated ISCs. ‡ Mostly dehydrated ISCs. ** P =0.01.

Kinetic studies of HbS polymer formation by turbidimetry of RBC lysatesfrom transplanted mice showed delayed HbS polymerization in lysates frommice transplanted with either SAD or BERK marrow transduced with eitherSAD or BERK marrow transduced with the β^(A-T87Q)-globin lentivirus(FIG. 5 c). The change in kinetics paralleled what was observed with RBClysates from homozygote SS patients versus asymptomatic ASheterozygotes.

The density of RBCs from transplanted SCD mouse models were examinedsince HbS polymerization causes abnormally high cell density (Blouin etal., supra; Nagel et al., supra, 2001). Whereas RBCs from control andmock-transduced SAD mice had a higher density than those of syngeneicC57BL/6 mice, mice completely reconstituted with β^(A-T87Q)-globinlentivirus-transduced SAD marrow showed a clear shift toward normal(FIG. 5 d). In BERK RBCs, the phenomenon was reversed, because theassociated thalassemic phenotype decreases the mean corpuscular Hbconcentration, resulting in lower cell density. The addition ofβ^(A-T87Q)-globin partially cured the thalassemia and resulted in highercell density, as shown in FIG. 5 d.

Unlike SAD mice, BERK mice have major alterations of their hematologicalparameters, as a consequence of both SCD and the associated thalassemia(Nagel et al., supra, 2001; Paszty et al., supra). In mice transplantedwith β^(A-T87Q)-globin lentivirus-transduced BERK marrow, RBC andreticulocyte counts were corrected with amelioration of Hbconcentration, anisocytosis, and poikilocytosis (Table 1).

In Vivo Analysis of SCD-Associated Symptoms

The amelioration of SCD-associated splenomegaly and characteristic urineconcentration defect in BERK mice was examined using gene therapy.Following transplantation of β^(A-T87Q)-globin lentivirus-transducedBERK marrow, both pathological features were corrected, whereas noeffect was observed for recipients of mock-transduced BERK marrow (Table1 and FIG. 5 e).

Example IV Analysis of Position Effect Variegation

In order to determine whether the observed pancellular expression ofhuman A-T87Q-globin protein in RBCs was the result ofposition-independent expression of the transferred A-T87Q-globin gene,bone marrow from a representative primary C57B1/6 recipient sacrificed 5months post-transplantation was used to generate day 12 spleen coloniesin secondary recipient mice. Southern blot analysis was performed ongenomic DNA isolated from 20 individual spleen colonies followingdigestion with BamH I, which cuts only once within the integratedprovirus. Ten distinct clones were observed with an average proviralcopy number of 3.4±0.3 (SE) (range 2-5). Primer extension analysis ofmRNA from spleen colonies showed a wide variation in the amount of humanA-T87Q-globin mRNA. These results suggest that pancellular expressionwas the result of balanced expression from polyclonal stem cellreconstitution with multiple chromosomal integration sites rather thantrue position-independent expression.

In summary, the previous examples demonstrate that chromosomalintegration of an antisickling globin gene variant in HSCs can result inits pancellular, erythroid-specific expression at levels sufficientlyhigh to correct the main pathological features of SCD. The previousexamples also demonstrate that structural optimization of theβ^(A-T87Q)-globin gene/LCR lentivirus by recombination-mediated cassetteexchange and incorporation of the central polypurine tract-DNA flap ofHIV-1, resulted in very high viral titers yielding multiple events ofchromosomal integration per hematopoietic stem cell. This integrationled to balanced expression which was sufficiently high and homogenousenough to provide an overall protection similar to that observed inasymptomatic human AS heterozygotes.

Example V In Vivo Analysis of Gene Therapy In Thalassemia Mouse Model

In order to test the therapeutic efficacy of a lentiviral vector for thetreatment of other hemoglobinpathies, the lentiviral vector containingwild-type human β globin gene was injected into the bone marrow of THALmice, a murine model used for studying β thalassemia. THAL mice bear ahomozygous deletion of the mouse β major gene and manifest ahypochromic, microcytic anemia with considerable anisocytosis,poikilocytosis, reticulocytosis, the presence of inclusion bodies in ahigh proportion of their circulating RBCs, and abnormally dehydratederythrocytes (Skow et al. (1983) Cell 34:1043).

The lentiviral globin gene vector used in the experiments isschematically shown in FIG. 14A. This vector is based on the designpreviously described in detail above and proven successful for thecorrection of sickle cell disease, except that it now incorporates thewild-type human β globin gene. The lentiviral vector notably containsspecific segments of promoter and LCR of the gene of interest, i.e.,human β globin, and also incorporates the central polypurine tract/DNAflap of HIV-1.

Recombinant virus pseudotyped with vesicular stomatitis virusglycoprotein-G was produced and subsequently concentrated 1,000-fold bytwo rounds of ultracentrifugation as described above and in Pawliuk etal. (2001), supra. A lentiviral vector carrying the gene that encodesenhanced GFP driven by the elongation factor 1-cc promoter was alsogenerated and used as a control in some of the following experiments.The absence of replication competent virus was verified by mobilizationassay. Viral titers were determined functionally by quantitativeSouthern blot analysis of transduced NIH 3T3 cells with proviral copynumber controls.

As shown in FIG. 14A, the vector contains HIV LTR, HIV type-1 longterminal repeat; Ψ+, packaging signal; cPPT, central polypurinetract/DNA flap; RRE, Rev-responsive element; E, exon; WS, interveningsequence; βP, β globin promoter (from SnaBI to Cap site); HS,hypersensitive site; ppt, polypurine tract. The 3′β globin enhancer (upto downstream AvrII site), the 372-bp IVS2 deletion (indicated by thetriangle) and DNase I hypersensitive sites, HS2 (SmaI to XbaI), HS3(SacI to PvuII) and HS4 (StuI to SpeI) of the LCR.

The [βglobin gene/LCR] lentiviral vector was optimized for viral titersand β globin gene expression by choosing specific segments of the βglobin gene, its promoter, and the β-LCR on the basis of results oftransgenic mouse experiments with single integrated copies andrecombination-mediated cassette exchange (Bouhassira et al. (1997) Blood90:3332) at the same sites of chromosomal integration in erythroid celllines. In addition, the vector contains the HIV-1 central polypurinetract/DNA flap for increased transduction efficiency, as shown in FIG.14A. After transient production in 293T cells on cotransfection with aplasmid encoding the pseudotyping vesicular stomatitis virusglycoprotein-G envelope and subsequent concentration byultracentrifugation, the [βglobin gene/LCR] lentiviral vector reachedfunctional titers of 1.5×10⁹ infectious units/ml, as assessed bySouthern blot analysis of transduced NIH 3T3 cells with proviral copynumber controls. The titers achieved were only 5-fold lower than thoseobtained with a similar lentiviral vector containing only the GFP genedriven by the elongation factor 1-α promoter.

In sum, the lentiviral vector incorporated several elements to enableproduction of stable, high-titer virus including a central polypurinetract/DNA flap element and rev-responsive element of HIV. Afterconcentration, viral preparations with titers exceeding 10⁹/ml wereachieved with a vector carrying an unmodified human β globin gene andextensive regions of the locus control region (LCR) including elementsof HS2, 3, and 4 without any evidence of viral instability throughabnormal splicing. The subsequent infection of murine bone marrow cellsat high MOI resulted in essentially 100% gene transfer to repopulatingcells with multiple proviral integrations per transduced cell.

Persistent Panerythroid Expression of Lentivirus-Encoded Human β GlobinIn THAL Mice

In order to ensure that β globin gene expression was persistent inmurine RBCs, FACS analysis was performed. FIG. 14B shows the results ofFACs analysis, with an antibody specific for human β globin chain, ofperipheral blood RBCs of lethally irradiated THAL mice transplanted 7months previously with syngeneic THAL bone marrow cells transduced witheither the [β globin/LCR] or the control GFP lentiviral vector asdescribed above. The proportion of peripheral blood RBCs expressinghuman β globin was assessed by FACS after staining the cells with anantibody specific for human β globin. RBCs were from THAL micetransplanted 7 months previously with bone marrow exposed to the lenti-βglobin vector. The viral titer used in experiment 1 (E1), was about2×10⁸ infectious units/ml and in experiments 2 and 3 (E2, E3) was about1.5×10⁹ infectious units/ml. In all mouse recipients of cells transducedwith the high-titer [β globin/LCR] vector preparations, about 95% ofRBCs were positive for human β globin protein (FIG. 14B). The high-levelreconstitution obtained with the high viral titer preparations wasassociated with a mean proviral copy number of about 3 per transducedcell, as assessed by Southern blot analysis of bone marrow, thymus, andspleens with proviral copy number controls. Time-course FACS analysesshowed that reconstitution of the mice with human β globin⁺ RBCs wasrapid, with pancellular expression observed as early as 2 months aftertransplant, and stable thereafter, even in THAL mice secondarilytransplanted with marrow cells harvested from the primary THALrecipients (shown in FIG. 14C).

Similar transplantation experiments with a lower titer viral preparation(2×10⁸ infectious units/ml) resulted in a lesser proportion of RBCsexpressing human β globin, indicating incomplete transduction of donorHSCs and position effect variegation of cells with a single integratedcopy (FIG. 14B). In summary, the human β globin was successfully andpersistently expressed in murine RBCs using the [β globin gene/LCR]lentiviral vector.

In Vivo Ccorrection of α Globin Imbalance In THAL Mice Using LentiviralVector

Expression of therapeutic levels of human β globin in the THAL miceresulted in correction of the α globin protein imbalance in these mice.Human β globin protein constituted on average 32.4±4% (27-39%) of all βglobin chains in RBC lysates of THAL mice in which anemia was correctedby transplantation, which was further supported by the results ofisoelectric focusing analyses of blood samples from the transplantedTHAL mice. Quantification of human β^(A) was performed by HPLC accordingto standard protocols. An additional Hb species was documented in allcorrected mice, consistent with the presence of Hb tetramer containingtwo murine α and two human β globin chains. Isoelectric focusing of RBClysates from transplanted primary mice showing the expected species ofHb with two murine α and two human β chains. Lanes 1-4, primaryrecipients; lanes 5 and 6, secondary recipients of lenti-βglobin-transduced THAL bone marrow cells. Quantification of human βglobin RNA in peripheral blood cells by RNA protection assay showed upto 130% levels relative to total mouse α globin RNA. As previouslyobserved, human β globin protein levels represent only a fraction ofthose of the encoding RNA, presumably because of intrinsic differencesin association constants between mouse and human globin chains to formHb tetramers and/or differential competition for globin translationbetween human and mouse mRNA species (Alami et al. (1997) Blood CellsMol Dis 25:110).

The transplanted THAL mice also had complete clearance of the excess ofmembrane-bound α globin chains, which represent about 1% of RBCmembrane-associated proteins in normal mice (1.4±0.7% in corrected THALmice versus 15.3±1% in GFP transplanted THAL controls).

In Vivo Expression of Human β Globin In THAL Mice Corrects HematologicParameters And Abnormal RBC Morphology In Lentiviral Recipients

THAL mice showing pancellular erythroid expression of human β globinshowed a marked improvement in all RBC indices, including decreasedreticulocyte percentage, increased RBCs, increased hematocritpercentage, and increased Hb (FIG. 15). Compared with pretransplantvalues, significant elevations (P<0.001) occurred in RBC number (from7×10⁶ to 9.7×10⁶ ±0.9 per mm³), hematocrit (from 28 to 40±2.3%), and Hbconcentration (from 8 to 12.4±0.7 g/dl) with RBC levels rising to withinthe normal range (no significant difference compared with normal,unmanipulated B6 mice) and just under normal levels for the hematocritand Hb concentration. This correction of anemia was further reflected ina dramatic reduction in reticulocyte numbers from levels of >20% to3.4±0.8%, again reaching the normal range. No improvement occurred inany parameters when THAL mice were transplanted with bone marrowtransduced with a GFP control vector as shown in FIG. 15.

In addition, histologic analysis was performed on the lentiviral andcontrol THAL mice to further examine the morphology associated with theimproved hematologic parameters. Findings from morphologic examinationof blood smears were consistent with the observed hematologicalimprovements. Blood smears of experimental and control animalsdemonstrated that correction of the anemia characteristic of THAL micewas associated with a marked reduction in RBC anisocytosis,poikilocytosis, and polychromasia. More than 75% of the RBCs werenormochromic and normocytic in both primary and secondary recipients.Control mice transplanted with lenti-GFP virus-transduced bone marrowcells remained severely anemic with marked reticulocytosis andmaintained their abnormal red cell morphology.

To assess further the correction of the anemia, RBC density analyseswere performed. Thalassemic RBCs have a decreased mean hemoglobinconcentration and correspondingly lower density as compared with normalRBCs (Fabry et al (1984) (, supra). In THAL recipients of [β globingene/LCR] lentivirus-transduced marrow, the density of RBCs narroweddramatically toward the normal range. In sum, phenotypic changes in RBCsand decrease in hemosiderin accumulation was observed in the spleen andliver of transplanted THAL mice.

Reversal of Thalassemia Disease Phenotype In Human β Globin TransducedRecipient THAL Mice

Recipients of [β globin gene/LCR] lentivirus-transduced bone marrowcells showed further evidence of a marked improvement in ineffectiveerythropoiesis. Spleen weight for B6, Thal/GFP, andThal/Lenti-β^(A)-treated mice was 110, 610, and 110 mg, respectively. Anincrease in the number of mature RBCs was observed in the red pulp ofthe spleens of Thal/Lenti-v^(A) mice as compared with unmanipulated THALmice. Moreover, the mature/immature nucleated red cell ratio was reducedfrom 20:80 in unmanipulated Thal mice to 40:60 in Thal/Lenti-β^(A) mice.These values corresponded well with the proportion of circulatingnormochromic normocytic red cells. The red pulp in theLenti-β^(A)-treated mice was only moderately expanded, compared with theconspicuous expansion of the red pulp in control THAL mice.

Untreated THAL mice showed significant extramedullary erythropoiesis inthe liver, whereas in the corrected THAL mice, extramedullaryerythropoiesis was mild to moderate. No erythropoiesis was observed inthe livers of normal B6 mice. Perls iron staining showed decreased ironaccumulation in the recipients of [β globin gene/LCR] lentivirustransduced marrow, thus providing further evidence of reduceddestruction of RBCs and improved erythropoiesis. The Perls iron stainingwas markedly reduced in the spleen and was negligible in the liver ofthe transplanted mice. In contrast, THAL mice had pronouncedaccumulation of hemosiderin in both the spleen and the liver. Normal B6mice had mild hemosiderin only in the spleen, and the liver wasnegative.

In summary, panerythroid permanent expression of the human β globin genewas observed in all THAL mice transplanted with [β globin gene/LCR]lentivirus transduced marrow, wherein the transplanted mice exhibited analmost complete correction of all observable disease manifestations.These experiments demonstrate that a lentiviral-based vector can deliveran expression cassette for human β globin, wherein the expression ofhuman β globin results in persistent, long-term correction ofineffective erythropoiesis and nearly complete cure of thalassemicphenotype in a mouse model for β-thalassemia. This correction waspossible in the absence of preselection for transduced cells beforetransplantation and was associated with essentially completereconstitution by genetically modified stem cells that resulted instable, high-level, pancellular expression of the human β globin gene inerythroid cells. These results constitute a significant advance overresults previously obtained using retroviral-based gene deliverysystems.

Example VI Construction of Self-Inactivating (SIN) Vector

To improve safety, the lentiviral vector described in the previousExamples was modified to include an insulator element in the right (3′)LTR as follows and as shown in FIGS. 6-13. First, modifications to the3′ (right) LTR of the vector were done by subcloning the 3′ LTR into aPuc19 plasmid using the Kpn 1 and Eco R1 sites, as shown in FIG. 7. A399 bp deletion was made in the U3 region of the right LTR (from the EcoRV site to the Pvu 2 site). The R region of the 3′ LTR was not altered.The U5 region was replaced with an ideal polyA sequence by digesting theLTR plasmid with Hind 3 and Sal 1 (FIG. 6). This removed a small portionof the R and U5 region. This region of the R and U5 regions was replacedwith the end portion of R and an ideal polyA sequence, thereby removingthe U5 region without changing the R region (FIG. 6). The followingoligo (OPLB 12/13,) was used to achieve this replacement:

(SEQ ID NO: 1) 5′-AAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTC GAC-3′wherein AAGCTT is the Hind 3 restriction site, GCCTTGAGTGCTTCA is theend of the R region, ATGTGTGTGTTGGTTTTTTGTGTG is the poly A sequence,and GTCGAC denotes the Sal 1 restriction site.

SIN vectors, which contain a chicken β-Globin insulator (cHS4), weremade by blunt end ligation into the EcoRV/Pvu II U3 deletion (see FIGS.6 and 9). Examples of the insulator sequences used include a 250 bpdoublet insulator (inserted into the U3 by blunting the Sal 1 and Not 1sites) (FIG. 6), and a 42 bp version of the insulator (inserted into toU3 deletion using an oligo (OHPV 460/461) which had engineered Cla 1 andPvu II sites) (FIG. 9). The modified 3′ LTR was then inserted back intothe vector using the Kpn 1 site and by blunting the EcoR1 site in thevector to the Sal 1 site from the plasmid containing the modified rightLTR. Two versions of the insulated SIN vector containing either GFP orβ-Globin were made (see FIGS. 7, 8, 10, and 11). Using the GFP insulatedSIN vector (250 bp doublet), concentrated virus was made with a titer of9.5×10⁹ TU/ml on 3T3 cells. FIG. 13 c shows that the substitution of theright U5 by a stronger poly(A) signal allows incorporation of cHS4insulator with a minimal decrease in viral titers.

Incorporation by Reference

The contents of all references and patents cited herein are herebyincorporated by reference in their entirety.

Equivalents

Although the invention has been described with reference to itspreferred embodiments, other embodiments can achieve the same results.Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, numerous equivalents to the specificembodiments described herein. Such equivalents are considered to bewithin the scope of this invention and are encompassed by the followingclaims.

1-31. (canceled)
 32. A self-inactivating (SIN) lentiviral vectorcomprising: a) a 5′ long terminal repeat (LTR); b) an RNA exportelement; c) a β-globin promoter; d) a β-globin locus control region(LCR); and e) a modified 3′ LTR comprising: i) at least one insulatorelement; or ii) a poly (A) sequence; wherein the β-globin promoter andβ-globin LCR are operatively linked to a gene of interest.
 33. Thevector of claim 32, wherein the 5′ LTR comprises a deletion compared tothe wild-type 5′ LTR and a heterologous promoter.
 34. The vector ofclaim 33, wherein the heterologous promoter is a cytomegalovirus (CMV)promoter.
 35. The vector of claim 32, wherein the RNA export elementcomprises a hepatitis B virus post-transcriptional regulatory element(PRE) or a human immunodeficiency virus (HIV) rev response element(RRE).
 36. The vector of claim 32, comprising a lentiviral centralpolypurine tract or DNA FLAP (cPPT/FLAP).
 37. The vector of claim 32,wherein the β-globin LCR comprises DNase I hypersensitive sites 2, 3,and 4 from the human β-globin LCR.
 38. The vector of claim 32,comprising a human β-globin 3′ enhancer element.
 39. The vector of claim32, comprising the at least one insulator element and the poly (A)sequence.
 40. The vector of claim 32, wherein the modified 3′ LTRcomprises two insulator elements.
 41. The vector of claim 32 or claim40, comprising an insulator sequence as set forth in SEQ ID NO:
 2. 42.The vector of claim 32 or claim 40, comprising an insulator sequence asset forth in nucleotides 8-49 of SEQ ID NO:
 2. 43. The vector of claim32, wherein the lentivirus is selected from the group consisting of:human immunodeficiency virus type 1 (HIV-1), human immunodeficiencyvirus type 2 (HIV-2), caprine arthritis-encephalitis virus (CAEV),equine infectious anemia virus (EIAV), feline immunodeficiency virus(FIV), bovine immune deficiency virus (BIV), and simian immunodeficiencyvirus (SIV).
 44. The vector of claim 32, wherein the modified 3′ LTRcomprises at least one deletion compared to the wild-type 3′ LTR. 45.The vector of claim 44, comprising the at least one insulator elementand the poly (A) sequence.
 46. The vector of claim 45, wherein themodified 3′ LTR comprises two insulator elements.
 47. The vector of anyone of claims 44-46, comprising an insulator sequence set forth in SEQID NO:
 2. 48. The vector of any one of claims 44-46, comprising aninsulator sequence as set forth in nucleotides 8-49 of SEQ ID NO:
 2. 49.The vector of claim 32, wherein the gene of interest encodes anantisickling protein or a globin gene.
 50. The vector of claim 32,wherein the gene of interest encodes a human β-globin gene, a humanδ-globin gene, or a human β^(A-T87Q)-globin gene.
 51. The vector ofclaim 32, comprising a nucleic acid cassette comprising a suicide geneoperably linked to a promoter or a gene for in vivo selection of thecell.
 52. The vector of claim 51, wherein the suicide gene is HSVthymidine kinase (HSV-Tk).
 53. The vector of claim 51, wherein the genefor in vivo selection is methylguanine methyltransferase (MGMT).
 54. Acell transduced with the vector of claim
 32. 55. The transduced cell ofclaim 54, wherein the cell is an embryonic stem cell, a somatic stemcell, or a progenitor cell.
 56. The transduced cell of claim 55, whereinthe cell is a bone marrow cell, a hematopoietic stem cell, or ahematopoietic progenitor cell.
 57. The transduced cell of claim 55,wherein the cell is an erythrocyte.
 58. A method of transplantingtransduced cells to a subject having a hemoglobinopathy comprisingadministering the transduced cells of claim 54 to the subject.
 59. Themethod of claim 58, wherein the transduced cells express atherapeutically effective amount of a human β-globin gene, a humanδ-globin gene, or a human β^(A-T87Q)-globin gene in the subject.
 60. Themethod of claim 58, wherein the hemoglobinopathy is selected from thegroup consisting of: hemoglobin sickle cell disease (SCD), sickle cellanemia, and β-thalassemia.